Abstract

This phase I clinical trial conducted with patients who had recurrent or progressive malignant glioma (MG) was designed to determine the maximum tolerated dose (MTD) and toxicity of three different 5-day dosing regimens of temozolomide (TMZ) in combination with O6-benzylguanine (O6-BG). Both TMZ and O6-BG were administered on days 1–5 of a 28-day treatment cycle. A bolus infusion of O6-BG was administered at 120 mg/m2 over 1 h on days 1, 3, and 5, along with a continuous infusion of O6-BG at 30 mg/m2/day. TMZ was administered at the end of the first bolus infusion of O6-BG and then every 24 h for 5 days during the continuous infusion of O6-BG. Patients were accrued to one of three 5-day dosing regimens of TMZ. Twenty-nine patients were enrolled into this study. The dose-limiting toxicities (DLTs) were grade 4 neutropenia, leukopenia, and thrombocytopenia. The MTD for TMZ for the three different 5-day dosing schedules was determined as follows: schedule 1, 200 mg/m2 on day 1 and 50 mg/m2/day on days 2–5; schedule 2, 50 mg/m2/day on days 1–5; and schedule 3, 50 mg/m2/day on days 1–5 while receiving pegfilgrastim. Thus, the 5-day TMZ dosing schedule that maximized the total dose of TMZ when combined with O6-BG was schedule 1. This study provides the foundation for a phase II trial of O6-BG in combination with a 5-day dosing schedule of TMZ in TMZ-resistant MG.

Gliomas are the most common type of primary malignant brain tumor, with poor survival despite conventional therapeutic regimens. The overall median survival from diagnosis for patients with malignant glioma (MG) is 48.2 weeks.1 Currently, the only two chemotherapeutics approved by the FDA for the treatment of MG, temozolomide (TMZ) and carmustine, both exert their therapeutic effect through alkylation. Unfortunately, the majority of patients with MG treated with alkylators, including TMZ, demonstrate de novo or acquired resistance, with subsequent tumor progression. Thus, targeting these mechanisms of resistance may maximize potential therapeutic benefit of these alkylating agents.

A major mechanism for resistance to alkylating chemotherapeutics is mediated in part through a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT).2–4 AGT removes chlorethylation or methylation damage from the O6 position of DNA guanines before cell injury and cell death. O6-benzylguanine (O6-BG) is a nonreversible inhibitor of the enzyme AGT and has been found to enhance cytotoxicity of alkylating chemotherapies, such as TMZ, in vitro2,5,6 and in in vivo xenografts.7,8 Thus, circumventing resistance and restoring chemotherapy sensitivity by combining O6-BG with TMZ may be a reasonable therapeutic strategy.

To develop such a therapeutic strategy, we must first determine the most efficacious and least toxic dosing regimen of TMZ and O6-BG when administered together. Although in vitro studies suggest that a 5-day dosing schedule of TMZ when combined with the O6-BG may be more efficacious than a 1-day dosing schedule, in vivo studies have been far less convincing. An in vitro study by Wedge et al.9 found the potentiation of TMZ cytotoxicity by O6-BG to increase linearly on 5 consecutive days. However, when Wedge et al.10 explored the effect of single versus multiple administration of O6-BG/TMZ combination in a human melanoma xenograft model, the results were not as convincingly supportive of the 5-day regimen over the 1-day regimen. Although Wedge et al.10 concluded that prolonged administration of the combination can lead to an increase in the therapeutic index of TMZ, no direct comparison of the 5-day to the 1-day regimen of TMZ plus O6-BG was made to support this conclusion. In fact, the efficacy and toxicity of these two regimens looked remarkably similar in the mice that received a single dose of TMZ at 200 mg/kg pretreated with 35 mg/kg O6-BG and the mice that received a total dose of 200 mg/kg of TMZ over 5 days pretreated each day with 35 mg/kg O6-BG. However, the major motivation for evaluating a 5-day versus a 1-day schedule of TMZ in combination with O6-BG was that the initial phase I trial of TMZ showed activity only in patients treated with the 5-day regimen,11 which led to this regimen being used in the subsequent phase II trials of TMZ.

We first explored the 1-day dosing schedule of TMZ in combination with O6-BG before exploring the 5-day dosing schedule of TMZ. Thus, we performed a phase I clinical trial in patients with MG to determine the maximum tolerated dose (MTD) of single-dose TMZ in combination with O6-BG.12 The MTD was 472mg/m 2 when TMZ was combined with O6-BG, a finding confirmed by Broniscer et al.13 In our phase II trial, we determined that O6-BG when added to a 1-day dosing regimen of TMZ was able to restore TMZ sensitivity in patients with TMZ-resistant anaplastic glioma, but no significant restoration of TMZ sensitivity was seen in patients with TMZ-resistant glioblastoma multiforme (GBM).14

Given the limited response in GBM on the 1-day dosing regimen of TMZ and O6-BG, we decided to explore the 5-day dosing regimen of TMZ in combination with O6-BG. However, before a 5-day dosing regimen of TMZ could be investigated, further clarification of the optimum dosing regimen of O6-BG in combination with the 5-day dosing regimen of TMZ was needed.

In our phase I and II trials utilizing the 1-day dosing regimen of TMZ,12,14 we chose an O6-BG dose of 120 mg/m2 over 1 h followed by a continuous infusion of 30 mg/m2/day for 48 h. This O6-BG dosing regimen was considered to be optimal for two reasons. First, a preclinical study by Hirose et al.15 found that the maximal TMZ sensitization in gliomas through prolonged AGT depletion with continuous O6-BG exposure was achieved 2–3 days after TMZ exposure. Second, Weingart et al.16 showed in a phase I trial that this dosing regimen suppressed tumor AGT for at least 48 h. Given the results in both of these studies, we considered the optimum O6-BG regimen for a 5-day dosing schedule of TMZ to be one where tumor AGT suppression occurred with certainty for at least 5 days, with 7 days perhaps being preferable. Thus, in order to guarantee tumor AGT suppression for at least 5 days, we chose to repeat the O6-BG regimen used in our phase I and II trial on days 1, 3, and 5.

Because no one knows the optimum dosing regimen of TMZ when combined with O6-BG, we chose to investigate three different 5-day regimens. Schedule 1 escalated the TMZ dose on days 2–5 but used a fixed dose of TMZ at 200 mg/kg on day 1. The reason for this uncustomary dosing regimen was our desire to incorporate the fact that one patient who participated in our prior phase I trial12 sustained a complete response while receiving the 1-day dosing regimen of TMZ at 200 mg/m2 along with O6-BG. This patient remained without tumor progression for 6 years and 4 months despite initially failing TMZ. Unlike schedule 1, schedule 2 escalated equal doses of TMZ for all 5 days. Schedule 3, while replicating the dosing schedule of schedule 2, incorporated pegfilgrastim to determine whether the MTD could be escalated beyond the limits found in schedule 2.

We now report a phase I clinical trial conducted in patients with recurrent or progressive MG. The primary objectives of the trial were to determine the safety and MTD of three different 5-day dosing regimens of TMZ in combination with O6-BG. The secondary objective was to provide a preliminary assessment of response.

Patients and Methods

Patient Population

Eligible patients had a histologically confirmed diagnosis of progressive or recurrent MG. They must have had measurable disease on contrast-enhanced MRI or CT scan (for patients when MRI was medically contraindicated) performed within 2 weeks of study drug administration. Patients were >18 years old and required to have a Karnofsky performance scale (KPS) score of >70%. An interval of at least 2 weeks since prior surgical resection, 4 weeks since prior chemotherapy (6 weeks for a nitrosourea-based regimen), or 12 weeks from prior radiotherapy had to have elapsed for the patient to be enrolled into the clinical trial. Additional enrollment criteria included adequate pretreatment bone marrow function (total granulocyte count >2,000 cells/μl, platelets >125,000 cells/μl), renal function (serum creatinine <1.5 upper limit of normal), and hepatic function (serum glutamic oxalacetic transaminase and bilirubin ≤ 2.5 times upper limit of normal). Patients of reproductive potential were required to take effective contraceptive measures for the duration of the study and for 2 months after completing therapy. All patients were informed of the investigational nature of the study and were required to sign an informed consent form as approved by the Duke University Health System Institutional Review Board.

The following patients were excluded from the study: pregnant or breast-feeding women, potentially fertile women or men who were not using an effective contraception method, patients treated in the past with the combination of TMZ plus O6-BG, patients with active infection requiring intravenous antibiotics, patients with a concurrently active primary malignancy, patients with known human immunodeficiency virus infection, and patients who received investigational drugs ≤ 2 weeks prior to starting on the study drug or who had not recovered from side effects of such therapy.

Study Design and Treatment

This was a phase I, open-label, single-center trial with accrual goals defined for three different dosing schedules of TMZ (Table 1). Schedule 1 consisted of a fixed dose of TMZ on day 1 at 200 mg/m2 and an initial dose of 25 mg/m2/day on days 2–5. The dose on days 2–5 was escalated for each new dose level by 25 mg/m2/day to determine the MTD. Schedule 2 consisted of the same dose of temozolomide on days 1–5. The starting dose level was 25 mg/m2/day higher than the dose found to be the MTD on days 2–5 for schedule 1. This dose was planned to be escalated by 25 mg/m2/day in subsequent cycles to determine the MTD. Schedule 3 started with a dose level 25 mg/m2/day higher than the MTD from schedule 2, and patients received pegfilgrastim between days 7 and 14 of each cycle. Dose escalation was planned to proceed by 25 mg/m2/day for each close level to determine the MTD.

Both TMZ and O6-BG were administered on days 1–5 of a 28-day treatment cycle. A 1-h bolus infusion of O6-BG was administered at 120 mg/m2 on day 1 and every 48 h on days 3 and 5. A continuous infusion of O6-BG at 30 mg/m2/day was administered immediately following the initial bolus infusion of O6-BG on day 1 and continued until immediately prior to the last bolus infusion. TMZ was administered orally, within 60 min of the end of the first bolus infusion of O6-BG (120 mg/m2) and then every 24 h for 5 days during the continuous infusion of O6-BG (30 mg/m2/day).

TMZ was commercially available as Temodar from Schering-Plough Research Institute (Kenilworth, NJ, USA). Pegfilgrastim was commercially available as Neulasta from Amgen (Thousand Oaks, CA, USA). O6-BG was supplied by AOI Pharmaceuticals, Inc. (New York, NY, USA).

Surveillance and Follow-up

The baseline examination included central review of tumor tissue, MRI or CT (if MRI was medically contraindicated), complete blood counts (CBCs) and blood chemistry tests, and a physical examination, including a comprehensive neurologic examination. During therapy, weekly CBCs were obtained. Prior to subsequent cycles of chemotherapy, patients were required to repeat CBC and blood chemistry tests, a physical examination, and neuroimaging.

Toxicity was graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 3.0 (http://ctep.cancer.gov/reporting/ctc_v30.html). Dose-limiting toxicity (DLT) must have occurred during the first cycle of therapy and was defined as the following: grade >4 neutropenia for >4 days, grade >3 febrile neutropenia (absolute neutrophil count <1 × 109/L, fever >38.5°C), grade >4 thrombocytopenia, grade >3 nonhematologic toxicity that is related to study drug (except grade >3 diarrhea without maximal intensive loperamide support, or grade >3 nausea or vomiting without maximal antiemetic therapy). Any adverse event causing >14 days of interruption to therapy (ie, a delay beyond day 42) would be considered a DLT, and the patient would be taken off study. The TMZ dose was reduced by 25 mg/m2/day if the patient developed a DLT. Patients who experienced recurrence of a DLT were taken off study.

Repeat cycles of chemotherapy were administered on schedule only if the patient met the following retreatment criteria: total granulocyte count >1,000 cells/μl; platelets >100,000/μl; serum glutamic oxalacetic transaminase, total bilirubin, and creatinine ≤ 2.5 times upper limit of normal; and all other toxicities resolved to baseline or grade 1.

Objective assessments of overall response were based on tumor assessment from MRI scans (CT if MRI was medically contraindicated) interpreted in light of corticosteroid use, as suggested by Macdonald et al.,17 with appropriate support from the neurologic examination.

This drug combination was administered for a maximum of 12 months or until unacceptable toxicity or tumor progression occurred. If the patient demonstrated ongoing radiographic and/or clinical improvement after 12 months of therapy, then the patient could continue therapy until there was evidence that maximal benefit had been achieved.

Statistical Analysis

In establishing the optimal TMZ dose when it was combined with O6-BG, a modified classic “3 + 3” dose-escalation design was employed, which permitted up to three additional patients to be accrued at a given dose level as long as none of the first three patients enrolled at that dose level experienced a DLT. The dose level was escalated in successive cohorts of three patients as long as no DLT was observed. If one instance of DLT was observed among the initial three assessable patients, an additional three patients had to be treated at that dose level with no further DLT in order for dose escalation to proceed. If two instances of DLT were observed at a dose level, the MTD was determined to be surpassed, and a total of six patients were treated at the previous level to ensure its tolerability. The MTD was therefore the highest dose that caused DLT in no more than one of six patients at that dose level. Any patient who had stable or responding disease that developed a DLT could continue to be treated at the next lowest dose level, provided the patient’s toxicity resolved to grade 1 or lower and no more than 2 weeks were required for recovery. However, the patient was removed from study if DLT occurred on the lower dose.

Results

Patient Characteristics

Between September 23, 2005, and April 10, 2006, a total of 29 patients with recurrent or progressive MG (WHO grades III and IV) were enrolled into the study at one of three dosing schedules for TMZ. Demographic and clinical characteristics of patients are detailed in Table 2.

Toxicity Evaluation

Of the 29 patients enrolled into the study, 28 were assessable for toxicity (Table 3). On schedule 1, at a TMZ dose of 200 mg/m2 on day 1 and 25 mg/m2/day on days 2–5, all five patients were assessable for toxicity, none of whom experienced a DLT. On schedule 1 at a TMZ dose of 200 mg/m2 on day 1 and 50 mg/m2/day on days 2–5, six of seven patients were assessable for toxicity. The patient that was not assessable for toxicity was lost to follow-up after study enrollment. One of the six patients experienced grade 4 neutropenia, which was dose limiting, and another patient experienced grade 3 thrombocytopenia, which was not dose limiting. On schedule 1 at a TMZ dose of 200 mg/m2 on day 1 and 75 mg/m2/day on days 2–5, all five patients were assessable for toxicity. Two patients experienced a DLT: one patient developed grade 4 leukopenia with grade 4 thrombocytopenia, and another patient developed grade 4 neutropenia with grade 3 thrombocytopenia. Two patients developed grade 3 neutropenia, which was not dose limiting. On schedule 2 at a TMZ dose of 75mg/m2/day on days 1–5, all six patients were assessable for toxicity. Two patients experienced a DLT: one patient developed grade 4 neutropenia with grade 4 thrombocytopenia, and another patient developed grade 4 neutropenia. One patient experienced grade 3 neutropenia, which was not dose limiting. On schedule 3 at a TMZ dose of 75 mg/m2/day on days 1–5 while receiving pegfilgrastim, all six patients were assessable for toxicity. Two patients experienced grade 4 neutropenia, which was dose limiting, and one patient experienced grade 3 neutropenia with grade 3 thrombocytopenia, which was not dose limiting. Thus, after the MTD was exceeded at a TMZ dose of 75 mg/m2/day for all three schedules, the MTD was established at 50 mg/m2/day. Note that this was inferred for schedules 2 and 3 since no patients on these schedules were treated at 50 mg/m2/day.

Response

All 29 patients who enrolled into the study were assessable for response. Seventeen patients showed disease progression after one cycle of chemotherapy. Three patients had stable disease for three cycles, and seven patients had stable disease for two cycles. Two patients had a partial response. One patient who responded was enrolled in schedule 2 and received TMZ at 75 mg/m2/day on days 1–5 for all 12 cycles. Even though this patient originally failed TMZ, the patient demonstrated a sustained response to TMZ and O6-BG for 14 months. Another patient who responded was enrolled in schedule 3 and received TMZ at 75 mg/m2/day on days 1–5 with pegfilgrastim but required a dose reduction to 50 mg/m2/day on days 1–5 because of grade 4 neutropenia. Although this patient also originally failed TMZ, the patient demonstrated a sustained response to TMZ and O6-BG for seven cycles.

Discussion

The results of this trial demonstrate that when TMZ is combined with O6-BG, the MTD for TMZ for three different 5-day dosing schedules was as follows: schedule 1 at a dose of 200 mg/m2 on day 1 and 50 mg/m2/day on days 2–5, schedule 2 at a dose of 50 mg/m2/day on days 1–5, and schedule 3 at a dose of 50 mg/m2/day on days 1–5 while receiving pegfilgrastim. Thus, the 5-day TMZ dosing schedule that maximizes the total dose of TMZ when combined with O6-BG is schedule 1, with TMZ administered at 200 mg/kg on day 1 and 50 mg/m2/day on days 2–5. DLT was limited to myelosuppression. Severe but reversible neutropenia, leukopenia, and thrombocytopenia were observed at TMZ doses of 50 and 75 mg/m2. Despite prior TMZ failure, two patients demonstrated a sustained response for 7 and 14 months. Subsequent studies will need to address toleration of repeated cycles of the MTD, information not readily available in this trial.

Although other O6-BG regimens have been used in combination with TMZ and polifeprosan 20 with carmustine implant (Gliadel), there is no evidence to suggest that these dosing regimens are adequate to suppress AGT levels in brain tumors. In a phase I trial after insertion of Gliadel, Weingart et al.16 attempted to deplete tumor AGT for up to 14 days using an O6-BG dosing regimen consisting of 120 mg/m2 over 1 h followed by a continuous infusion of 30 mg/m2/day for up to 14 days. Unfortunately, examination of the pharmacokinetics for the prolonged continuous infusion of O6-BG raised the possibility that a higher dose of O6-BG will be needed in future studies to suppress tumor AGT activity for the full 2 weeks. Another alternative O6-BG dosing regimen used in a phase I trial after treatment with a 5-day dosing regimen of TMZ utilized a daily 1-h infusion O6-BG at 120 mg/m2 without a continuous infusion.18 Although research shows depletion of AGT at 18 h after this 1-h infusion of O6-BG, it is highly unlikely and has never been proven that this depletion is continuous from 18 to 24 h, which makes this a less attractive alternative.

The phase I trial of O6-BG and TMZ reported here has defined the therapeutic approach for our ongoing phase II trial of TMZ plus O6-BG, which will enroll patients with recurrent TMZ-resistant MG. This will provide the evidence needed to determine whether TMZ plus O6-BG can restore TMZ sensitivity in patients for whom this agent has previously failed. Other strategies to overcome TMZ resistance mediated by AGT include a dose-dense TMZ schedule, an approach currently being evaluated by the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer.19,20

Acknowledgments

This study was supported by National Institute of Neurological Disorders and Stroke grant 5P50 NS20023-25, National Institutes of Health (NIH) Specialized Programs of Research Excellence grant 5P50 CA108786-4, and NIH merit award R37 CA 011898-38. S.X.J. was supported by NIH grant TL1 RR024126.